Schneider et al. BMC Microbiology 2018, 18(Suppl 1):140 https://doi.org/10.1186/s12866-018-1291-8 RESEARCH Open Access High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.) Daniela I Schneider1,2, Andrew G Parker3, Adly M Abd-alla3 and Wolfgang J Miller1* Abstract Background: In African tsetse flies Glossina, spp. detection of bacterial symbionts such as Wolbachia is challenging since their prevalence and distribution are patchy, and natural symbiont titers can range at levels far below detection limit of standard molecular techniques. Reliable estimation of symbiont infection frequency, especially with regard to interrelations between symbionts and their potential impact on host biology, is of pivotal interest in the context of future applications for the control and eradication of Glossina-vectored African trypanosomosis. The presence or absence of symbionts is routinely screened with endpoint polymerase chain reaction (PCR), which has numerous advantages, but reaches its limits, when detecting infections at natural low titer. To not only determine presence of native tsetse symbionts but also to localize them to specific host tissues, fluorescence in situ hybridization (FISH) can be applied. However, classic FISH assays may not detect low-titer infections due to limitations in sensitivity. Results: We have compared classic endpoint PCR with high-sensitivity blot-PCR. We demonstrate that the latter technique allows for clear detection of low-titer Wolbachia in the morsitans and palpalis groups while classic endpoint PCR does not. In order to localize Wolbachia in situ in high and low-titer Glossina species, weappliedhigh-endStellaris®rRNA-FISH. We show that with this high sensitivity method, even low amounts of Wolbachia can be traced in specific tissues. Furthermore, we highlight that more tissues and organs than previously recorded are infested with Wolbachia in subspecies of the morsitans and palpalis groups. Conclusions: Our results demonstrate that overall symbiont infection frequencies as well as the presence in specific host tissues may be underestimated when using low-sensitivity methods. To better understand the complex interrelation of tsetse flies and their native symbionts plus the pathogenic trypanosomes, it is important to consider application of a broader range of high-sensitivity detection tools. Keywords: Wolbachia, Low-titer symbiont detection limit, Stellaris® fluorescence in situ hybridization, Tissue tropism Background eradicate African trypanosomosis in the future [4]. In the African tsetse fly (Glossina spp., Diptera: Glossini- While classic endpoint PCR has numerous advantages, it dae), detection of bacterial symbionts such as Wolbachia soon reaches its limits, when it comes to detecting very is challenging since their prevalence and distribution are low loads of symbionts (low-titer infections), and localize patchy [1], and their natural titers can range at levels far the symbionts to specific host tissues (tissue tropism). below detection limit of standard molecular techniques Classic fluorescence in situ hybridization (FISH) using [2, 3]. Reliable estimation of symbiont infection fre- 16S rRNA oligo probes is the method of choice for fol- quency, however, especially with regard to interrelations lowing tissue tropism. However, sensitivity may be too between the symbionts and their potential impact on low for symbiont detection in biologically relevant host host biology, is of pivotal interest in order to control and organs like testes but also for the in situ detection of generally low-titer infections such as the ones of G. p. * Correspondence: [email protected] gambiensis. In the presented study, we have compared 1Department Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria the feasibility of classic PCR and blot-PCR for low-titer Full list of author information is available at the end of the article Wolbachia in species and subspecies of the genus © International Atomic Energy Agency; licensee BioMed Central Ltd. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution IGO License (https://creativecommons.org/licenses/by/3.0/igo/) which permits unrestricted use, distribution, and reproduction in any medium, provided appropriate credit to the original author(s) and the source is given. Schneider et al. BMC Microbiology 2018, 18(Suppl 1):140 Page 148 of 292 Glossina. Furthermore, we took advantage of the novel Stellaris® technology to trace such low-titer Wolbachia in situ. As known from recent studies, not only high- titer infections, but also Wolbachia low-titer infections impact host biology [5]. Hence, their reliable detection Fig. 1 Detection of Wolbachia in Glossina females and males via with particular regard to their location in situ, is import- polymerase chain reaction (PCR). a Wolbachia-specific single copy ant for better understanding host-symbiont relations wsp-PCR detects the symbiont only in high-titer Gmm (♀, ♂) and plus the crosstalk with trypanosome parasites. Gmc (♀, ♂). b The more sensitive multicopy ARM-PCR additionally detects wGsw (Wolbachia of G. swynnertoni). In the Wolbachia- Results uninfected G. f. fuscipes, ARM-PCR amplifies a possibly nonspecific product of smaller size. Negative controls are Wolbachia-uninfected Low-titer Wolbachia infections in Glossina spp. may be minus Drosophila simulans, NouméaTC (Dsim ) and non-template overlooked with standard endpoint-PCR techniques control (NTC). Females are first on gel. Each PCR was at least To demonstrate the advantage of more sensitive detec- repeated once in order to confirm results. Abbreviations: wsp tion methods over standard techniques, we employed Wolbachia outer surface protein gene andcomparedtwoendpointPCRtechniquesplusone high-end blot-PCR. First, we tested the classic Wolba- wsp-PCR, the more sensitive multicopy specific ARM- chia marker wsp (Wolbachia outer surface protein PCR easily detects high-titer Wolbachia of Gmm and gene) with Glossina subspecies from the palpalis and Gmc. In addition, it traces the earlier described low-titer morsitans groups (Table 1). infection in Gsw [3]. Interestingly ARM-PCR successfully Endpoint PCR using the single copy wsp marker amplifies a smaller fragment in Gfu males but not fe- clearly detects high-titer Wolbachia in females and males, (Fig. 1b; and see below). males of G. m. morsitans (Gmm)andG. m. centralis (Gmc), but G. swynnertoni (Gsw) and G. p. gambiensis High-end blot-PCR increases Wolbachia detection limit (Gpg) seem uninfected. We did also not detect Wolba- As demonstrated above, wsp and ARM markers are effi- chia in the two subspecies G. p. palpalis (Gpp) and G. f. cient tools to screen for Wolbachia infection status fuscipes (Gfu), which were previously reported Wolba- when symbiont titers are at high or medium levels. In chia-negative (Fig. 1a)[1]. Next, we applied the multi- order to further increase the detection limit of our assay, copy locus ARM (Wolbachia A-supergroup repeat we employed a combined PCR-hybridization (blot-PCR) motif) [3] to the same sample set (Fig. 1b). Similar to assay [2, 6]. This method has proven reliable in detecting Table 1 List of Glossina and Drosophila strains analyzed in this study Species Species Strain/ Origin/reference group abbr. Drosophila Drosophila simulans simulans NouméaTC [18] Wolbachia negative strains control Drosophila willistoni willistoni P98 [19] Wolbachia positive control Wolbachia status (experimental) wsp ARM blot FISH Glossina strains Glossina morsitans morsitans morsitans Gmm Takáč Lab, Slovak Academy of Sciences, Bratislava, ++ ++ ++ ++ Slovakia + + + + Glossina morsitans centralis morsitans Gmc Insect Pest Control Laboratory FAO/IAEA, Vienna, ++ ++ ++ ++ Austria + + + + Glossina swynnertoni morsitans Gsw Insect Pest Control Laboratory FAO/IAEA, Vienna, – ++ ++ nd Austria Glossina palpalis palpalis palpalis Gpp Insect Pest Control Laboratory FAO/IAEA, Vienna, –––– Austria Glossina palpalis palpalis Gpg Insect Pest Control Laboratory FAO/IAEA, Vienna, ––++ gambiensis Austria Glossina fuscipes fuscipes palpalis Gfu Insect Pest Control Laboratory FAO/IAEA, Vienna, ––+? nd Austria Two Drosophila strains were used as Wolbachia-positive and -negative controls (P98, NouméaTC). The table lists all Glossina strains used for experiments, including Wolbachia infection status based on PCR (Wolbachia outer surface protein gene, Wolbachia A-supergroup repeat motif,); blot-PCR using a wsp probe (listed as ‘blot’), and fluorescence in situ hybridization with Wolbachia 16-23S rRNA probe. Wolbachia infection titer is indicated by ‘+’ (low), ‘++’ (intermediate), and ‘+++’ (high). Abbreviations: wsp Wolbachia outer surface protein gene, ARM Wolbachia A-supergroup repeat motif, FISH fluorescence in situ hybridization, nd not determined Schneider et al. BMC Microbiology 2018, 18(Suppl 1):140 Page 149 of 292 low loads of Wolbachia, which were undetected in although Wolbachia from Gmm and Gmc are well standard PCR assays such as the wsp-based PCR [2, 5, known for causing strong cytoplasmic incompatibility 6]. As shown in Fig. 2b consequent hybridization with (CI) [2, 8]. By using high-end Stellaris® rRNA-FISH, an internal Wolbachia-specific wsp-probe allows more however, we clearly detected the symbiont in the testes Wolbachia to be traced than in the wsp-PCR assay of both Gmm and Gmc. Most interestingly, we did not shown in Fig. 2a. Similar to classic endpoint wsp-PCR,